Means and methods for assessing liver enzyme induction

ABSTRACT

The present invention pertains to the field of toxicological assessments for risk stratification of chemical compounds. Specifically, it relates to a method for diagnosing the pro-pathological effect of compounds which are inducing liver enzymes. It also relates to a method of determining whether a compound is capable of exhibiting pro-pathological effects on the liver by enzyme induction in a subject and to a method of identifying a drug for treating the pro-pathological effect of liver enzyme induction. Furthermore, the present invention relates to a data collection comprising characteristic values of at least five analytes, a data storage medium comprising said data collection, and a system and a device for diagnosing the pro-pathological effect of liver enzyme induction. Finally, the present invention pertains to the use of a group of analytes or means for the determination thereof for the manufacture of a diagnostic device or composition for diagnosing the pro-pathological effect of liver enzyme induction in a subject. For each sex, a different metabolome pattern, i.e. a different set of analytes is disclosed. The liver enzyme induction markers are mainly selected from free fatty acids, but also include various phosphatidylcholines, galactose, 3- and 5-Methoxysphingosine, Cholesterol, Threonic acid, 1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine, Glycerol, Glycerophosphate, Dodecanol, myo-Inositol-2-monophosphate.

The present invention pertains to the field of toxicological assessmentsfor risk stratification of chemical compounds. Specifically, it relatesto a method for diagnosing liver enzyme induction. It also relates to amethod of determining whether a compound is capable of inducing suchliver enzyme induction in a subject and to a method of identifying adrug for treating liver enzyme induction. Furthermore, the presentinvention relates to a data collection comprising characteristic valuesof at least five analytes, a data storage medium comprising said datacollection, and a system and a device for diagnosing liver enzymeinduction. Finally, the present invention pertains to the use of a groupof analytes or means for the determination thereof for the manufactureof a diagnostic device or composition for diagnosing liver enzymeinduction in a subject.

The major functions of the liver include metabolic functions,detoxification and bile excretion. These functions are carried out byliver enzymes. Depending on the requirements for metabolic enzymes ordetoxification enzymes, the activity of these enzymes will increase ordecrease in the liver. The activity increase of the liver enzymes inresponse to an increased requirement is called enzyme induction. Enzymeinduction may lead to a potentially pro-pathological condition,especially as far as the detoxification of other compounds is concerned.Also there are numerous examples, particularly of carcinogens, whichrequire metabolic activation before they can exert a carcinogeniceffect. Thus, enzyme induction can potentially increase the formation ofsuch activated compounds.

Enzyme induction could be seen as an increased predisposition fordeveloping liver toxicity and accompanying diseases or disorders. Livertoxicity may become apparent in a subject by various disorders, diseasesor medical conditions including liver cell necrosis, hepatitis,steatosis, cirrhosis, phospholipoidosis, cholestasis, cholangitis (seee.g., Grunhagen 2003, Z. Gastroenterol. 41(6): 565-578).

Enzyme induction can be observed in response to various chemicalcompounds to which the liver will be exposed. Among these compounds,there are voluntarily exposed chemical compounds such as drugs ornutritional compounds contained by the food as well as chemicalcompounds taken up inevitably from the environment. Contrary to thechemical compounds which exhibit a direct toxic liver effect, compoundsmay merely result in enzyme induction which per se does not becomeapparent as a pathological condition but which increases the risk for asubject for developing pathological liver toxicity or any of theaforementioned liver associated diseases.

As a consequence of the said pro-pathological condition, care should betaken not to expose the liver to other factors which in combination withthe pro-pathological enzyme induction may result in liver toxicity or anassociated disease.

The current methods for determining enzyme induction do not allow for areliable diagnosis on the potential for liver toxicity effects of thecompounds (see e.g. G. G. Gibson and P. Skett 2001, Introduction to DrugMetabolism, Chapter 3: Induction and Inhibition of Drug Metabolism,3^(rd) Edition, Nelson Thornes Publishers, Cheltenham, UK; E. M. Bomhardet al. 1998, Toxicology 131:73-91; A. Lahoz et al. 2008, Current DrugMetabolism 9:12-19). The importance of enzyme induction and a potentialsubsequent liver toxicity, however, may become apparent if one considersthat liver toxicity by now is the most common reason for a drug to bewithdrawn from the market.

Moreover, chemical compounds which are used in any kind of industry inthe European Community, e.g., will now need to comply with REACH(Registration, Evaluation and Authorisation of Chemicals). It will beunderstood that the potential of a chemical compound to induce livertoxicity will be deemed as a high risk for the compound and,consequently, the compound will be available only for limitedapplications and when obeying high security standards.

Sensitive and specific methods for assessing the toxicologicalproperties of a chemical compound which induces liver enzymes in anefficient and reliable manner are not yet available but would,nevertheless, be highly appreciated.

Thus, the technical problem underlying the present invention could beseen as the provision of means and methods for complying with theaforementioned needs. The technical problem is solved by the embodimentscharacterized in the claims and described herein below.

Accordingly, the present invention relates to a method for diagnosingliver enzyme induction comprising:

-   -   (a) determining the amount of at least one, preferably, at least        five of the following analytes Stearic acid (C18:0), Galactose,        Lignoceric acid (C24:0), Behenic acid (C22:0), Nervonic acid        (C24:1), 3- and 5-Methoxysphingosine, Cholesterol, Threonic        acid, Phosphatidylcholine (C18:0/C18:2),        1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine, Eicosapentanoic        acid, Phosphatidylcholine (C18:0/C22:6) in a test sample of a        male subject suspected to show liver enzyme induction or at        least one, preferably, at least five of the following analytes        Glycerol, Palmitic acid (C16:0), Linoleic acid (C18:cis[9,12]2),        Stearic acid (C18:0), Arachidonic acid (C20:cis-[5,8,11,14]4),        Docosahexaenoic acid (C22:cis[4,7,10,13,16,19]6), Cholesterol,        Glycerolphosphate, Galactose, Lignoceric acid (C24:0),        Dodecanol, Heptadecanoic acid (C17:0), Eicosanoic acid (C20:0),        myo-Inositol-2-monophosphate, Behenic acid (C22:0), Nervonic        acid (C24:1), gamma-Linolenic acid (C18:cis[6,9,12]3), 3- and        5-Methoxysphingosine, Threonic acid, Phosphatidylcholine (C18:2,        C20:4), Phosphatidylcholine (C18:0/C18:2) in a test sample of a        female subject suspected to show liver enzyme induction, and    -   (b) comparing the amounts determined in step (a) to a reference,        whereby liver enzyme induction is to be diagnosed.

The term “Phosphatidylcholine (C18:0/C18:2)” as used herein refers tomolecular species, preferably, characterized by the sum parameter ofglycerophosphorylcholines containing the combination of a C18:0 fattyacid unit and a C18:2 fatty acid unit. The mass-to-charge ratio (m/z) ofthe ionised spezies is 786.6 Da (+/−0.3 Da). A preferred fragmentationpattern is shown in FIG. 1, below.

The term “1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine” as usedherein refers to molecular species, preferably, characterized by the sumparameter of glycerophosphorylcholines containing fatty acid units withthe total number of carbons of 36 and the total number of double bondsof 1. The mass-to-charge ratio (m/z) of the ionised spezies is 788.6 Da(+/−0.3 Da).

The term “Phosphatidylcholine (C18:0/C22:6)” as used herein refers tomolecular species, preferably, characterized by the sum parameter ofglycerophosphorylcholines containing the combination of a C18:0 fattyacid unit and a C22:6 fatty acid unit. The mass-to-charge ratio (m/z) ofthe ionised spezies is 834.6 Da (+/−0.3 Da). A preferred fragmentationpattern is shown in FIG. 2, below.

The expression “method for diagnosing” as referred to in accordance withthe present invention means that the method either essentially consistsof the aforementioned steps or may include further steps. However, it isto be understood that the method, in a preferred embodiment, is a methodcarried out ex vivo, i.e. not practised on the human or animal body.Diagnosing as used herein refers to assessing the probability accordingto which a subject is suffering from a disease. As will be understood bythose skilled in the art, such an assessment, although preferred to be,may usually not be correct for 100% of the subjects to be diagnosed. Theterm, however, requires that a statistically significant portion ofsubjects can be identified as suffering from the disease or as having apredisposition therefor. Whether a portion is statistically significantcan be determined without further ado by the person skilled in the artusing various well known statistic evaluation tools, e.g., determinationof confidence intervals, p-value determination, WELCH-test, Mann-Whitneytest, etc. Details are found in Dowdy and Wearden, Statistics forResearch, John Wiley & Sons, New York 1983. Preferred confidenceintervals are at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%. The p-values are, preferably, 0.2, 0.1, 0.05.

Diagnosing according to the present invention includes monitoring,confirmation, and classification of the relevant disease or itssymptoms. Monitoring relates to keeping track of an already diagnoseddisease, e.g. to analyze the progression of the disease, the influenceof a particular treatment on the progression of disease or complicationsarising during the disease period or after successful treatment of thedisease. Confirmation relates to the strengthening or substantiating adiagnosis already performed using other indicators or markers.

Classification relates to allocating the diagnosis according to thestrength or kind of symptoms into different classes. Some of thediseases or conditions which appear in connection with liver enzymeinduction may be accompanied by further metabolic changes. Moreover,classification also, preferably, includes allocating a mode of action toa compound to be tested by the method of the present invention.Specifically, the method of the present invention allows fordetermination of a specific mode of action of a compound for which suchmode of action is not yet known. This is, preferably, achieved bycomparing the determined analytes representing biomarkers (i.e. thebiomarker profile) for said compound to the biomarker profile of acompound for which the mode of action is known as a reference. Theclassification of the mode of action allows an even more reliableassessment of the enzyme induction properties of a compound because themolecular targets of the compound are identified. Due to saididentification, further parameters influencing liver enzyme inductionmay be taken into account. The term “liver enzyme induction” or “enzymeinduction” as used herein relates to a pro-pathological conditionwherein metabolic and detoxification enzymes in the liver aresignificantly increased with respect to activity and abundance comparedto the upper limits of normal found in a population. More preferably,the induced enzymes include at least cytochrome P450 enzymes,alkoxyresorufin-O-Deethylase,4-hydroxybiphenyl-UDP-glucuronosyltransferase and/or4-methylumbelliferone-UDP-glucuronosyltransferase. Preferably, liverenzyme induction is a predisposition for liver toxicity and diseases ordisorder accompanied therewith, more preferably, liver cell necrosis,hepatitis, steatosis, cirrhosis, phospholipoidosis, cholestasis,cholangitis. The symptoms and clinical signs of the aforementioneddiseases and disorders are well known to the person skilled in the artand are described in detail H. Marquardt, S. G. Schäfer, R. O.McClellan, F. Welsch (eds.), “Toxicology”, Chapter 13: The Liver, 1999,Academic Press, London.

Each of the analytes to be determined in the method of the presentinvention is also suitable for diagnosing the diseases or disordersreferred to herein when analysed alone. However, it was found inaccordance with the present invention that a combination of at leastfive different analytes further strengthen the diagnosis since each ofthe analytes is an apparently statistically independent predictor ofequal value for the diagnosis. Moreover, the specificity for livertoxicity is also significantly increased since influences from othertissues on the marker abundance are counterbalanced. Preferred markercombinations for specific liver enzyme inducing compound classes arethose found in Table 1 and 2, below.

It is to be understood that in addition to a group consisting of atleast five of the aforementioned analytes, additional analytes are,preferably, determined in the method of the present invention. Theadditional analytes are, preferably, also selected from theaforementioned group. In other words, preferably, at least six, at leastseven, at least eight, at least nine or all of the analytes of theaforementioned group are determined in the method of the presentinvention. The additional determination of these analytes even furtherstrengthens the result obtained by the method of this invention.Furthermore, other analytes or metabolites (i.e. metabolites notspecifically recited in the aforementioned group) or biomarkers, such asenzymes, may still be determined in addition. Preferably, additionalparameters determined in accordance with the method of the presentinvention are: total cytochrome P450 content,alkoxyresorufin-O-Deethylase,4-hydroxybiphenyl-UDP-glucuronosyltransferase or4-methylumbelliferone-UDP-glucuronosyltransferase.

In a particular preferred embodiment, the at least one analyte in themale sample is selected from the group consisting of: Stearic acid(C18:0), Lignoceric acid (C24:0), Behenic acid (C22:0), Cholesterol, andEicosapentanoic acid. In an even more preferred embodiments, all of theaforementioned analytes are determined.

However, it is also preferably envisaged that in a group of at leastfive analytes to be determined in accordance with the present invention,one, two, three, or four analytes are from the aforementioned group ofpreferred analytes while the remaining analytes are analytes for malesamples as specified elsewhere herein.

Thus, if the first analyte of a preferred group of five analytes to bedetermined in accordance with the present invention is Stearic acid(C18:0), the remaining four analytes are selected from a groupconsisting of the following analytes: Galactose, Lignoceric acid(C24:0), Behenic acid (C22:0), Nervonic acid (C24:1), 3- and5-Methoxysphingosine, Cholesterol, Threonic acid, Phosphatidylcholine(C18:0/C18:2), 1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine,Eicosapentanoic acid, Phosphatidyl-choline (C18:0/C22:6).

If the first and second analyte are Stearic acid (C18:0) and Lignocericacid (C24:0), the remaining three analytes are selected from a groupconsisting of: Galactose, Behenic acid (C22:0), Nervonic acid (C24:1),3- and 5-Methoxysphingosine, Cholesterol, Threonic acid,Phosphatidylcholine (C18:0/C18:2),1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine, Eicosapentanoic acid,Phosphatidylcholine (C18:0/C22:6).

If the first, second and third analytes are Stearic acid (C18:0),Lignoceric acid (C24:0), and Behenic acid (C22:0), the remaining twoanalytes are selected from a group consisting of: Galactose, Nervonicacid (C24:1), 3- and 5-Methoxysphingosine, Cholesterol, Threonic acid,Phosphatidylcholine (C18:0/C18:2),1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine, Eicosapentanoic acid,Phosphatidylcholine (C18:0/C22:6).

If the first, second, third and fourth analytes are Stearic acid(C18:0), Lignoceric acid (C24:0), Behenic acid (C22:0), and Cholesterol,the remaining analyte is selected from a group consisting of: Galactose,Nervonic acid (C24:1), 3- and 5-Methoxysphingosine, Threonic acid,Phosphatidylcholine (C18:0/C18:2),1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine, Eicosapentanoic acid,Phosphatidylcholine (C18:0/C22:6).

In a particular preferred embodiment, the at least one analyte in thefemale is selected from the group consisting of: Glycerol, Palmitic acid(C16:0), Linoleic acid (C18:cis[9,12]2), Stearic acid (C18:0),Arachidonic acid (C20:cis-[5,8,11,14]4), Cholesterol, Lignoceric acid(C24:0), Eicosanoic acid (C20:0), and Behenic acid (C22:0). In an evenmore preferred embodiments, all six of the aforementioned analytes aredetermined. However, it is preferably envisaged that in a group of atleast five analytes to be determined in accordance with the presentinvention, one, two, three, or four analytes are from the aforementionedgroup of preferred analytes while the remaining analytes are analytesfor female samples as specified elsewhere herein.

However, it is also preferably envisaged that in a group of at leastfive analytes to be determined in accordance with the present invention,one, two, three, or four analytes are from the aforementioned group ofpreferred analytes while the remaining analytes are analytes for malesamples as specified elsewhere herein.

Thus, if the first analyte of a preferred group of five analytes to bedetermined in accordance with the present invention is Glycerol, theremaining four analytes are selected from a group consisting of thefollowing analytes: Palmitic acid (C16:0), Linoleic acid(C18:cis[9,12]2), Stearic acid (C18:0), Arachidonic acid(C20:cis-[5,8,11,14]4), Docosahexaenoic acid(C22:cis[4,7,10,13,16,19]6), Cholesterol, Glycerolphosphate, Galactose,Lignoceric acid (C24:0), Dodecanol, Heptadecanoic acid (C17:0),Eicosanoic acid (C20:0), myo-Inositol-2-monophosphate, Behenic acid(C22:0), Nervonic acid (C24:1), gamma-Linolenic acid (C18:cis[6,9,12]3),3- and 5-Methoxysphingosine, Threonic acid, Phosphatidylcholine (C18:2,C20:4), Phosphatidylcholine (C18:0/C18:2).

If the first and second analyte are Glycerol and Palmitic acid (C16:0),the remaining three analytes are selected from a group consisting of:Linoleic acid (C18:cis[9,12]2), Stearic acid (C18:0), Arachidonic acid(C20:cis-[5,8,11,14]4), Docosahexaenoic acid(C22:cis[4,7,10,13,16,19]6), Cholesterol, Glycerolphosphate, Galactose,Lignoceric acid (C24:0), Dodecanol, Heptadecanoic acid (C17:0),Eicosanoic acid (C20:0), myo-Inositol-2-monophosphate, Behenic acid(C22:0), Nervonic acid (C24:1), gamma-Linolenic acid (C18:cis[6,9,12]3),3- and 5-Methoxysphingosine, Threonic acid, Phosphatidylcholine (C18:2,C20:4), Phosphatidylcholine (C18:0/C18:2).

If the first, second and third analytes are Glycerol, Palmitic acid(C16:0), and Linoleic acid (C18:cis[9,12]2), the remaining two analytesare selected from a group consisting of: Stearic acid (C18:0),Arachidonic acid (C20:cis-[5,8,11,14]4), Docosahexaenoic acid(C22:cis[4,7,10,13,16,19]6), Cholesterol, Glycerolphosphate, Galactose,Lignoceric acid (C24:0), Dodecanol, Heptadecanoic acid (C17:0),Eicosanoic acid (C20:0), myo-Inositol-2-monophosphate, Behenic acid(C22:0), Nervonic acid (C24:1), gamma-Linolenic acid (C18:cis[6,9,12]3),3- and 5-Methoxysphingosine, Threonic acid, Phosphatidylcholine (C18:2,C20:4), Phosphatidylcholine (C18:0/C18:2).

If the first, second, third and fourth analytes are Glycerol, Palmiticacid (C16:0), Linoleic acid (C18:cis[9,12]2), and Stearic acid (C18:0),the remaining analyte is selected from a group consisting of:Arachidonic acid (C20:cis-[5,8,11,14]4), Docosahexaenoic acid(C22:cis[4,7,10,13,16,19]6), Cholesterol, Glycerolphosphate, Galactose,Lignoceric acid (C24:0), Dodecanol, Heptadecanoic acid (C17:0),Eicosanoic acid (C20:0), myo-Inositol-2-monophosphate, Behenic acid(C22:0), Nervonic acid (C24:1), gamma-Linolenic acid (C18:cis[6,9,12]3),3- and 5-Methoxysphingosine, Threonic acid, Phosphatidylcholine (C18:2,C20:4), Phosphatidylcholine (C18:0/C18:2).

Analyte as used herein refers to at least one molecule of a specificanalyte up to a plurality of molecules of the said specific analyte. Itis to be understood further that a group of analytes means a pluralityof chemically different molecules wherein for each analyte at least onemolecule up to a plurality of molecules may be present. An analyte inaccordance with the present invention encompasses all classes of organicor inorganic chemical compounds including those being comprised bybiological material such as organisms. Preferably, the analyte inaccordance with the present invention is a small molecule compound. Morepreferably, in case a plurality of analytes is envisaged, it will beunderstood that each analyte represents a metabolite and that theplurality of metabolites represent a metabolome. The metabolome is thecollection of metabolites being comprised by an organism, an organ, atissue or a cell at a specific time and under specific conditions.

Metabolites are small molecule compounds, such as substrates for enzymesof metabolic pathways, intermediates of such pathways or the productsobtained by a metabolic pathway. Metabolic pathways are well known inthe art and may vary between species. Preferably, said pathways includeat least citric acid cycle, respiratory chain, glycolysis,gluconeogenesis, hexose monophosphate pathway, oxidative pentosephosphate pathway, production and β-oxidation of fatty acids, ureacycle, amino acid biosynthesis pathways, protein degradation pathwayssuch as proteasomal degradation, amino acid degrading pathways,biosynthesis or degradation of: lipids, polyketides (including e.g.flavonoids and isoflavonoids), isoprenoids (including eg. terpenes,sterols, steroids, carotenoids), carbohydrates, phenylpropanoids andderivatives, alcaloids, benzenoids, indoles, indole-sulfur compounds,porphyrines, hormones, vitamins, cofactors such as prosthetic groups orelectron carriers, glucosinolates, purines, pyrimidines, nucleosides,nucleotides and related molecules such as tRNAs, microRNAs (miRNA) ormRNAs. Accordingly, small molecule compound metabolites are preferablycomposed of the following classes of compounds: alcohols, alkanes,alkenes, alkines, aromatic compounds, ketones, aldehydes, carboxylicacids, esters, amines, imines, amides, cyanides, amino acids, peptides,thiols, thioesters, phosphate esters, sulfate esters, thioethers,sulfoxides, ethers, or combinations or derivatives of the aforementionedcompounds. The small molecules among the metabolites may be primarymetabolites which are required for normal cellular function, organfunction or animal growth, development or health. Moreover, smallmolecule metabolites further comprise secondary metabolites havingessential ecological function, e.g. metabolites which allow an organismto adapt to its environment. Furthermore, metabolites are not limited tosaid primary and secondary metabolites and further encompass artificialsmall molecule compounds. Said artificial small molecule compounds arederived from exogenously provided small molecules which are administeredor taken up by an organism but are not primary or secondary metabolitesas defined above. For instance, artificial small molecule compounds maybe metabolic products obtained from drugs by metabolic pathways of theanimal. Moreover, metabolites further include peptides, oligopeptides,polypeptides, oligonucleotides and polynucleotides, such as RNA or DNA.More preferably, a metabolite has a molecular weight of 50 Da (Dalton)to 30,000 Da, most preferably less than 30,000 Da, less than 20,000 Da,less than 15,000 Da, less than 10,000 Da, less than 8,000 Da, less than7,000 Da, less than 6,000 Da, less than 5,000 Da, less than 4,000 Da,less than 3,000 Da, less than 2,000 Da, less than 1,000 Da, less than500 Da, less than 300 Da, less than 200 Da, less than 100 Da.Preferably, a metabolite has, however, a molecular weight of at least 50Da. Most preferably, a metabolite in accordance with the presentinvention has a molecular weight of 50 Da up to 1,500 Da.

Analytes as referred to in accordance with this invention are molecularspecies which are derived from the naturally occurring metabolite due tothe purification and/or determination process. In some cases, theanalyte will be identical. In other cases, however, it will be achemical derivative thereof. Nevertheless, it is to be understood thatthe appearing of the analyte inevitably allows drawing conclusions onthe occurrence of the metabolite.

The term “test sample” as used herein refers to samples to be used forthe diagnosis of liver enzyme induction by the method of the presentinvention. Said test sample is a biological sample. Samples frombiological sources (i.e. biological samples) usually comprise aplurality of metabolites. Preferred biological samples to be used in themethod of the present invention are samples from body fluids,preferably, blood, plasma, serum, saliva, bile, urine or cerebrospinalfluid, or samples derived, e.g., by biopsy, from cells, tissues ororgans, preferably from the liver. More preferably, the sample is ablood, plasma or serum sample, most preferably, a plasma sample.Biological samples are derived from a subject as specified elsewhereherein. Techniques for obtaining the aforementioned different types ofbiological samples are well known in the art. For example, blood samplesmay be obtained by blood taking while tissue or organ samples are to beobtained, e.g., by biopsy.

The aforementioned samples are, preferably, pre-treated before they areused for the method of the present invention. As described in moredetail below, said pre-treatment may include treatments required torelease or separate the compounds or to remove excessive material orwaste. Suitable techniques comprise centrifugation, extraction,fractioning, ultrafiltration, protein precipitation followed byfiltration and purification and/or enrichment of compounds. Moreover,other pre-treatments are carried out in order to provide the compoundsin a form or concentration suitable for compound analysis. For example,if gas-chromatography coupled mass spectrometry is used in the method ofthe present invention, it will be required to derivatize the compoundsprior to the said gas chromatography. Suitable and necessarypre-treatments depend on the means used for carrying out the method ofthe invention and are well known to the person skilled in the art.Pre-treated samples as described before are also comprised by the term“sample” as used in accordance with the present invention.

The term “subject” as used herein relates to animals, preferably tomammals such as mice, rats, guinea pigs, rabbits, hamsters, pigs, sheep,dogs, cats, horses, monkeys, or cows and, also preferably, to humans.More preferably, the subject is a rodent and, most preferably, a rat.Other animals which may be diagnosed applying the method of the presentinvention are fishes, birds or reptiles. Preferably, said subject was inor has been brought into contact with a compound suspected to be capableof inducing liver enzymes. A subject which has been brought into contactwith a compound suspected to induce liver enzymes may, e.g., be alaboratory animal such as a rat which is used in a screening assay for,e.g., toxicity of compounds. A subject suspected to have been in contactwith a compound capable of inducing liver enzymes may be also a subjectto be diagnosed for selecting a suitable therapy. Preferably, a compoundcapable of inducing liver enzymes as used herein is Phenobarbitalsodium, Aroclor 1254, Pentachlorobenzene, Beta-ionone, Ethyl-benzene orVinclozoline.

The term “determining the amount” as used herein refers to determiningat least one characteristic feature of each analyte of the said at leastfive analytes. Characteristic features in accordance with the presentinvention are features which characterize the physical and/or chemicalproperties including biochemical properties of an analyte. Suchproperties include, e.g., molecular weight, viscosity, density,electrical charge, spin, optical activity, colour, fluorescence,chemoluminescence, elementary composition, chemical structure,capability to react with other compounds, capability to elicit aresponse in a biological read out system (e.g., induction of a reportergene) and the like. Values for said properties may serve ascharacteristic features and can be determined by techniques well knownin the art. Moreover, the characteristic feature may be any featurewhich is derived from the values of the physical and/or chemicalproperties of a analyte by standard operations, e.g., mathematicalcalculations such as multiplication, division or logarithmic calculus.Most preferably, the at least one characteristic feature allows thedetermination and/or chemical identification of the analyte and itsamount. Accordingly, the characteristic value, preferably, alsocomprises information relating to the abundance of the metabolite fromwhich the characteristic value is derived. For example, a characteristicvalue of a metabolite may be a peak in a mass spectrum. Such a peakcontains characteristic information of the metabolite, i.e. the m/z(mass to charge ratio) information, as well as an intensity value beingrelated to the abundance of the said analyte (i.e. its amount) in thesample.

As discussed before, each analyte of the group of analytes to bedetermined in accordance with the method of the present invention maybe, preferably, determined quantitatively or semi-quantitatively. Forquantitative determination, either the absolute or precise amount of theanalyte will be determined or the relative amount of the analyte will bedetermined based on the value determined for the characteristicfeature(s) referred to herein above. The relative amount may bedetermined in a case were the precise amount of a analyte can or shallnot be determined. In said case, it can be determined whether the amountin which the analyte is present is enlarged or diminished with respectto a second sample comprising said metabolite in a second amount.Quantitatively analysing a analyte, thus, also includes what issometimes referred to as semi-quantitative analysis of a analyte.

Moreover, determining as used in the method of the present invention,preferably, includes using a compound separation step prior to theanalysis step referred to before. Preferably, said compound separationstep yields a time resolved separation of the analytes comprised by thesample. Suitable techniques for separation to be used preferably inaccordance with the present invention, therefore, include allchromatographic separation techniques such as liquid chromatography(LC), high performance liquid chromatography (HPLC), gas chromatography(GC), thin layer chromatography, size exclusion or affinitychromatography. These techniques are well known in the art and can beapplied by the person skilled in the art without further ado. Mostpreferably, LC and/or GC are chromatographic techniques to be envisagedby the method of the present invention. Suitable devices for suchdetermination of analytes are well known in the art. Preferably, massspectrometry is used in particular gas chromatography mass spectrometry(GC-MS), liquid chromatography mass spectrometry (LC-MS), directinfusion mass spectrometry or Fourier transform ion-cyclotrone-resonancemass spectrometry (FT-ICR-MS), capillary electrophoresis massspectrometry (CE-MS), high-performance liquid chromatography coupledmass spectrometry (HPLC-MS), quadrupole mass spectrometry, anysequentially coupled mass spectrometry, such as MS-MS or MS-MS-MS,inductively coupled plasma mass spectrometry (ICP-MS), pyrolysis massspectrometry (Py-MS), ion mobility mass spectrometry or time of flightmass spectrometry (TOF). Most preferably, LC-MS and/or GC-MS are used asdescribed in detail below. Said techniques are disclosed in, e.g.,Nissen, Journal of Chromatography A, 703, 1995: 37-57, U.S. Pat. No.4,540,884 or U.S. Pat. No. 5,397,894, the disclosure content of which ishereby incorporated by reference. As an alternative or in addition tomass spectrometry techniques, the following techniques may be used forcompound determination: nuclear magnetic resonance (NMR), magneticresonance imaging (MRI), Fourier transform infrared analysis (FT-IR),ultraviolet (UV) spectroscopy, refraction index (RI), fluorescentdetection, radiochemical detection, electrochemical detection, lightscattering (LS), dispersive Raman spectroscopy or flame ionisationdetection (FID). These techniques are well known to the person skilledin the art and can be applied without further ado. The method of thepresent invention shall be, preferably, assisted by automation. Forexample, sample processing or pre-treatment can be automated byrobotics. Data processing and comparison is, preferably, assisted bysuitable computer programs and databases. Automation as described hereinbefore allows using the method of the present invention inhigh-throughput approaches.

Moreover, the analyte can also be determined by a specific chemical orbiological assay. Said assay shall comprise means which allow forspecifically detecting the analyte in the sample. Preferably, said meansare capable of specifically recognizing the chemical structure of theanalyte or are capable of specifically identifying the analyte based onits capability to react with other compounds or its capability to elicita response in a biological read out system (e.g., induction of areporter gene). Means which are capable of specifically recognizing thechemical structure of an analyte are, preferably, antibodies or otherproteins which specifically interact with chemical structures, such asreceptors or enzymes. Specific antibodies, for instance, may be obtainedusing the metabolite as antigen by methods well known in the art.Antibodies as referred to herein include both polyclonal and monoclonalantibodies, as well as fragments thereof, such as Fv, Fab and F(ab)₂fragments that are capable of binding the antigen or hapten. The presentinvention also includes humanized hybrid antibodies wherein amino acidsequences of a non-human donor antibody exhibiting a desiredantigen-specificity are combined with sequences of a human acceptorantibody. Moreover, encompassed are single chain antibodies. The donorsequences will usually include at least the antigen-binding amino acidresidues of the donor but may comprise other structurally and/orfunctionally relevant amino acid residues of the donor antibody as well.Such hybrids can be prepared by several methods well known in the art.Suitable proteins which are capable of specifically recognizing theanalyte are, preferably, enzymes which are involved in the metabolicconversion of the analyte or metabolite. Said enzymes may either use theanalyte as a substrate or may convert a substrate into the analyte.Moreover, said antibodies may be used as a basis to generateoligopeptides which specifically recognize the analyte. Theseoligopeptides shall, for example, comprise the enzyme's binding domainsor pockets for the said analyte. Suitable antibody and/or enzyme basedassays may be RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbentassay), sandwich enzyme immune tests, electrochemiluminescence sandwichimmunoassays (ECLIA), dissociation-enhanced lanthanide fluoro immunoassay (DELFIA) or solid phase immune tests. Moreover, the analyte mayalso be identified based on its capability to react with othercompounds, i.e. by a specific chemical reaction. Further, the metabolitemay be determined in a sample due to its capability to elicit a responsein a biological read out system. The biological response shall bedetected as read out indicating the presence and/or the amount of theanalyte comprised by the sample. The biological response may be, e.g.,the induction of gene expression or a phenotypic response of a cell oran organism.

The term “reference” refers to values of characteristic features of eachof the analytes of the group of analytes which can be correlated toliver enzyme induction. Such reference results are, preferably, obtainedfrom a sample derived from a subject which has been brought into contactwith Phenobarbital sodium, Aroclor 1254, Pentachlorobenzene,Beta-ionone, Ethyl-benzene or Vinclozoline. A subject may be broughtinto contact with the said compounds by each topic or systemicadministration mode as long as the compounds are bioavailable. Thereference results may be determined as described hereinabove for theamounts of the analytes. Alternatively, but nevertheless also preferred,the reference results may be obtained from sample derived from a subjectwhich has not been brought into contact with Phenobarbital sodium,Aroclor 1254, Pentachlorobenzene, Beta-ionone, Ethyl-benzene orVinclozoline or a healthy subject with respect to liver enzyme inductionand, more preferably, other diseases or disorders as well. Moreover, thereference, also preferably, could be a calculated reference, mostpreferably the average or median, for the relative or absolute amountfor each of the analytes of the group of analytes derived from apopulation of individuals comprising the subject to be investigated.However, it is to be understood that the population of subjects to beinvestigated for determining a calculated reference, preferably, eitherconsist of apparently healthy subjects (e.g. untreated) or comprise anumber of apparently healthy subjects which is large enough to bestatistically resistant against significant average or median changesdue to the presence of the test subject(s) in the said population. Theabsolute or relative amounts of the analytes of said individuals of thepopulation can be determined as specified elsewhere herein. How tocalculate a suitable reference value, preferably, the average or median,is well known in the art. The population of subjects referred to beforeshall comprise a plurality of subjects, preferably, at least 5, 10, 50,100, 1,000 or 10,000 subjects. It is to be understood that the subjectto be diagnosed by the method of the present invention and the subjectsof the said plurality of subjects are of the same species.

More preferably, the reference results, i.e. values for at least onecharacteristic features of the analyte, will be stored in a suitabledata storage medium such as a database and are, thus, also available forfuture diagnoses.

The term “comparing” refers to assessing whether the results of thedetermination described hereinabove in detail, i.e. the results of thequalitative or quantitative determination of an analyte, are identicalor similar to reference results or differ therefrom.

In case the reference results are obtained from a sample derived from asubject which has been brought into contact with Phenobarbital sodium,Aroclor 1254, Pentachlorobenzene, Beta-ionone, Ethyl-benzene orVinclozoline, liver enzyme induction can be diagnosed based on thedegree of identity or similarity between the test results obtained fromthe test sample and the aforementioned reference results, i.e. based onan identical or similar qualitative or quantitative composition withrespect to the aforementioned analytes. The results of the test sampleand the reference results are identical, if the values for thecharacteristic features and, in the case of quantitative determination,the intensity values are identical. Said results are similar, if thevalues of the characteristic features are identical but the intensityvalues are different. Such a difference is, preferably, not significantand shall be characterized in that the values for the intensity arewithin at least the interval between 1^(st) and 99^(th) percentile,5^(th) and 95^(th) percentile, 10^(th) and 90^(th) percentile, 20^(th)and 80^(th) percentile, 30^(th) and 70^(th) percentile, 40^(th) and60^(th) percentile of the reference value the 50^(th), 60^(th), 70^(th),80^(th), 90^(th) or 95^(th) percentile of the reference value.

In case the reference results are obtained a subject which has not beenbrought into contact with Phenobarbital sodium, Aroclor 1254,Pentachlorobenzene, Beta-ionone, Ethyl-benzene or Vinclozoline, liverenzyme induction can be diagnosed based on the differences between thetest results obtained from the test sample and the aforementionedreference results, i.e. differences in the qualitative or quantitativecomposition with respect to the aforementioned analytes. The sameapplies if a calculated reference as specified above is used. Thedifference may be an increase in the absolute or relative amount of ananalyte (sometimes referred to as up-regulation of the analyte; see alsoExamples) or a decrease in either of said amounts or the absence of adetectable amount of the analyte (sometimes referred to as up-regulationof the analyte; see also Examples). Preferably, the difference in therelative or absolute amount is significant, i.e. outside of the intervalbetween 45^(th) and 55^(th) percentile, 40^(th) and 60^(th) percentile,30^(th) and 70^(th) percentile, 20^(th) and 80^(th) percentile, 10^(th)and 90^(th) percentile, 5^(th) and 95^(th) percentile, 1^(st) and99^(th) percentile of the reference value.

Preferably, the amounts of the analytes in comparison to the referencediffer as follows: (i) in a sample of a male: Stearic acid (C18:0),Galactose, Lignoceric acid (C24:0), Behenic acid (C22:0), Nervonic acid(C24:1), 3- and 5-Methoxysphingosine, Cholesterol, Threonic acid,Phosphatidylcholine (C18:0/C18:2),1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine, Eicosapentanoic acid,Phosphatidylcholine (C18:0/C22:6) all of which are increased and (ii) ina sample of a female subject: Glycerol, Palmitic acid (C16:0), Linoleicacid (C18:cis[9,12]2), Stearic acid (C18:0), Arachidonic acid(C20:cis-[5,8,11,14]4), Docosahexaenoic acid(C22:cis[4,7,10,13,16,19]6), Cholesterol, Glycerolphosphate, Galactose,Lignoceric acid (C24:0), Dodecanol, Heptadecanoic acid (C17:0),Eicosanoic acid (C20:0), myo-Inositol-2-monophosphate, Behenic acid(C22:0), Nervonic acid (C24:1), gamma-Linolenic acid (C18:cis[6,9,12]3),3- and 5-Methoxysphingosine, Threonic acid, Phosphatidylcholine (C18:2,C20:4), Phosphatidylcholine (C18:0/C18:2) all of which are increased.For the specific analytes referred to in this specification, preferredvalues for the changes in the relative amounts (i.e. “fold”-changes) orthe kind of change (i.e. “up”- or “down”-regulation resulting in ahigher or lower relative and/or absolute amount) are indicated in theExamples below.

The comparison is, preferably, assisted by automation. For example, asuitable computer program comprising algorithm for the comparison of twodifferent data sets (e.g., data sets comprising the values of thecharacteristic feature(s)) may be used. Such computer programs andalgorithm are well known in the art. Notwithstanding the above, acomparison can also be carried out manually.

The aforementioned methods for the determination of the analytes can beimplemented into a device. A device as used herein shall comprise atleast the aforementioned means. Moreover, the device, preferably,further comprises means for comparison and evaluation of the detectedcharacteristic feature(s) of the analytes and, also preferably, thedetermined signal intensity. The means of the device are, preferably,operatively linked to each other. How to link the means in an operatingmanner will depend on the type of means included into the device. Forexample, where means for automatically qualitatively or quantitativelydetermining the metabolite are applied, the data obtained by saidautomatically operating means can be processed by, e.g., a computerprogram in order to facilitate the diagnosis. Preferably, the means arecomprised by a single device in such a case. Said device may accordinglyinclude an analyzing unit for the analytes and a computer unit forprocessing the resulting data for the diagnosis. Alternatively, wheremeans such as test stripes are used for determining the analytes, themeans for diagnosing may comprise control stripes or tables allocatingthe determined result data to result data known to be accompanied withliver enzyme induction or those being indicative for a healthy subjectas discussed above. Preferred devices are those which can be appliedwithout the particular knowledge of a specialized clinician, e.g., teststripes or electronic devices which merely require loading with asample.

Alternatively, the methods for the determination of the analytes can beimplemented into a system comprising several devices which are,preferably, operatively linked to each other. Specifically, the meansmust be linked in a manner as to allow carrying out the method of thepresent invention as described in detail above. Therefore, operativelylinked, as used herein, preferably, means functionally linked. Dependingon the means to be used for the system of the present invention, saidmeans may be functionally linked by connecting each mean with the otherby means which allow data transport in between said means, e.g., glassfiber cables, and other cables for high throughput data transport.Nevertheless, wireless data transfer between the means is also envisagedby the present invention, e.g., via LAN (Wireless LAN, W-LAN). Apreferred system comprises means for determining analytes. Means fordetermining analytes as used herein encompass means for separatinganalytes, such as chromatographic devices, and means for analytedetermination, such as mass spectrometry devices. Suitable devices havebeen described in detail above. Preferred means for compound separationto be used in the system of the present invention includechromatographic devices, more preferably devices for liquidchromatography, HPLC, and/or gas chromatography. Preferred devices forcompound determination comprise mass spectrometry devices, morepreferably, GC-MS, LC-MS, direct infusion mass spectrometry, FT-ICR-MS,CE-MS, HPLC-MS, quadrupole mass spectrometry, sequentially coupled massspectrometry (including MS-MS or MS-MS-MS), ICP-MS, Py-MS or TOF. Theseparation and determination means are, preferably, coupled to eachother. Most preferably, LC-MS and/or GC-MS is used in the system of thepresent invention as described in detail elsewhere in the specification.Further comprised shall be means for comparing and/or analyzing theresults obtained from the means for determination of analytes. The meansfor comparing and/or analyzing the results may comprise at least onedatabases and an implemented computer program for comparison of theresults. Preferred embodiments of the aforementioned systems and devicesare also described in detail below.

Advantageously, it has been found in the study underlying the presentinvention that the amounts of a group of at least five of theaforementioned analytes serve as biomarkers for liver enzyme inductionand, thus, a predisposition for liver toxicity. The specificity andaccuracy of the method will be even more improved by determining all ofthe aforementioned analytes. A change in the quantitative and/orqualitative composition of the metabolome with respect to these specificanalytes is indicative for liver enzyme induction. The morphological,physiological as well as biochemical parameters which are currently usedfor diagnosing liver enzyme induction are less specific and lesssensitive in comparison to the biomarker determination provided by thepresent invention. Thanks to the present invention, liver enzymeinduction properties of a compound can be more efficiently and reliablyassessed. Moreover, based on the aforementioned findings, screeningassays for drugs which are useful for the therapy or amelioration ofliver enzyme induction are feasible.

The present invention, in principle, relates to the use of at least one,preferably, at least five of the following analytes Stearic acid(C18:0), Galactose, Lignoceric acid (C24:0), Behenic acid (C22:0),Nervonic acid (C24:1), 3- and 5-Methoxysphingosine, Cholesterol,Threonic acid, Phosphatidylcholine (C18:0/C18:2),1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine, Eicosapentanoic acid,Phosphatidylcholine (C18:0/C22:6) in a sample of a male subject and/orat least one, preferably, at least five of the following analytesGlycerol, Palmitic acid (C16:0), Linoleic acid (C18:cis[9,12]2), Stearicacid (C18:0), Arachidonic acid (C20:cis-[5,8,11,14]4), Docosahexaenoicacid (C22:cis[4,7,10,13,16,19]6), Cholesterol, Glycerolphosphate,Galactose, Lignoceric acid (C24:0), Dodecanol, Heptadecanoic acid(C17:0), Eicosanoic acid (C20:0), myo-Inositol-2-monophosphate, Behenicacid (C22:0), Nervonic acid (C24:1), gamma-Linolenic acid(C18:cis[6,9,12]3), 3- and 5-Methoxysphingosine, Threonic acid,Phosphatidylcholine (C18:2, C20:4), Phosphatidylcholine (C18:0/C18:2) ina sample of a female subject or means for the detection thereof for themanufacture of a diagnostic device or composition for diagnosing liverenzyme induction.

All definitions and explanations of the terms made above apply mutatismutandis for the aforementioned methods and all other embodimentsdescribed further below except stated otherwise in the following.

It follows from the above that the present invention also contemplates amethod of determining whether a compound is capable of inducing liverenzyme induction in a subject comprising:

-   -   (a) determining in a sample of a male subject which has been        brought into contact with a compound suspected to be capable of        inducing liver enzyme induction the amount of at least one,        preferably, at least five of the following analytes Stearic acid        (C18:0), Galactose, Lignoceric acid (C24:0), Behenic acid        (C22:0), Nervonic acid (C24:1), 3- and 5-Methoxysphingosine,        Cholesterol, Threonic acid, Phosphatidylcholine (C18:0/C18:2),        1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine, Eicosapentanoic        acid, Phosphatidylcholine (C18:0/C22:6) or in a test sample of a        female which has been brought into contact with a compound        suspected to be capable of inducing liver enzyme induction at        least one, preferably, at least five of the following analytes        Glycerol, Palmitic acid (C16:0), Linoleic acid (C18:cis[9,12]2),        Stearic acid (C18:0), Arachidonic acid (C20:cis-[5,8,11,14]4),        Docosahexaenoic acid (C22:cis[4,7,10,13,16,19]6), Cholesterol,        Glycerolphosphate, Galactose, Lignoceric acid (C24:0),        Dodecanol, Heptadecanoic acid (C17:0), Eicosanoic acid (C20:0),        myo-Inositol-2-monophosphate, Behenic acid (C22:0), Nervonic        acid (C24:1), gamma-Linolenic acid (C18:cis[6,9,12]3), 3- and        5-Methoxysphingosine, Threonic acid, Phosphatidylcholine (C18:2,        C20:4), Phosphatidylcholine (C18:0/C18:2); and    -   (b) comparing the amounts determined in step (a) to a reference,        whereby the capability of the compound to induce liver enzyme        induction is determined.

In a preferred embodiment of said method, said compound is at least onecompound selected from the group consisting of: Phenobarbital sodium,Aroclor 1254, Pentachlorobenzene, Beta-ionone, Ethyl-benzene orVinclozoline.

Preferably, said reference is derived from a subject which suffers fromliver enzyme induction or from a subject which has been brought intocontact with at least one compound selected from the group consistingof: Phenobarbital sodium, Aroclor 1254, Pentachlorobenzene, Beta-ionone,Ethyl-benzene or Vinclozoline. More preferably, essentially identicalamounts for the analytes in the test sample and the reference areindicative for liver enzyme induction.

Also preferably, said reference is derived from a subject known to notsuffer from liver enzyme induction or from a subject which has not beenbrought into contact with at least one compound selected from the groupconsisting of: Phenobarbital sodium, Aroclor 1254, Pentachlorobenzene,Beta-ionone, Ethyl-benzene or Vinclozoline. Alternatively, but alsopreferred, the said reference is a calculated reference for the analytesfor a population of subjects. More preferably, amounts for the analyteswhich differ in the test sample in comparison to the reference areindicative for liver enzyme induction.

Preferred indicative amounts for liver enzyme induction are disclosedelsewhere in this specification.

The present invention also relates to a method of identifying asubstance for treating liver enzyme induction comprising the steps of:

-   -   (a) determining in a sample of a male subject suffering from        liver enzyme induction which has been brought into contact with        a candidate substance suspected to be capable of treating liver        enzyme induction the amount of at least one, preferably, at        least five of the following analytes Stearic acid (C18:0),        Galactose, Lignoceric acid (C24:0), Behenic acid (C22:0),        Nervonic acid (C24:1), 3- and 5-Methoxysphingosine, Cholesterol,        Threonic acid, Phosphatidylcholine (C18:0/C18:2),        1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine, Eicosapentanoic        acid, Phosphatidylcholine (C18:0/C22:6) or in a sample of a        female subject suffering from liver enzyme induction which has        been brought into contact with a candidate substance suspected        to be capable of treating liver enzyme induction the amount of        at least one, preferably, at least five of the following        analytes Glycerol, Palmitic acid (C16:0), Linoleic acid        (C18:cis[9,12]2), Stearic acid (C18:0), Arachidonic acid        (C20:cis-[5,8,11,14]4), Docosahexaenoic acid        (C22:cis[4,7,10,13,16,19]6), Cholesterol, Glycerolphosphate,        Galactose, Lignoceric acid (C24:0), Dodecanol, Heptadecanoic        acid (C17:0), Eicosanoic acid (C20:0),        myo-Inositol-2-monophosphate, Behenic acid (C22:0), Nervonic        acid (C24:1), gamma-Linolenic acid (C18:cis[6,9,12]3), 3- and        5-Methoxysphingosine, Threonic acid, Phosphatidylcholine (C18:2,        C20:4), Phosphatidylcholine (C18:0/C18:2); and    -   (b) comparing the amounts determined in step (a) to a reference,        whereby a substance capable of treating liver enzyme induction        is to be identified.

Specifically, in case of the method of identifying a substance usefulfor treating liver enzyme induction, said reference is, preferably,derived from a subject which has been brought into contact with at leastone compound selected from the group consisting of: Phenobarbitalsodium, Aroclor 1254, Pentachlorobenzene, Beta-ionone, Ethyl-benzene orVinclozoline or a subject which suffers from liver enzyme induction.More preferably, amounts for the analytes which differ in the testsample and the reference are indicative for a substance useful fortreating liver enzyme induction.

Specifically, indicative for a substance capable of treating liverenzyme induction are amounts of the analytes in comparison to thereference which differ as follows: (i) in a sample of a male: Stearicacid (C18:0), Galactose, Lignoceric acid (C24:0), Behenic acid (C22:0),Nervonic acid (C24:1), 3- and 5-Methoxysphingosine, Cholesterol,Threonic acid, Phosphatidylcholine (C18:0/C18:2),1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine, Eicosapentanoic acid,Phosphatidylcholine (C18:0/C22:6) all of which are decreased and (ii) ina sample of a female subject: Glycerol, Palmitic acid (C16:0), Linoleicacid (C18:cis[9,12]2), Stearic acid (C18:0), Arachidonic acid(C20:cis-[5,8,11,14]4), Docosahexaenoic acid(C22:cis[4,7,10,13,16,19]6), Cholesterol, Glycerolphosphate, Galactose,Lignoceric acid (C24:0), Dodecanol, Heptadecanoic acid (C17:0),Eicosanoic acid (C20:0), myo-Inositol-2-monophosphate, Behenic acid(C22:0), Nervonic acid (C24:1), gamma-Linolenic acid (C18:cis[6,9,12]3),3- and 5-Methoxysphingosine, Threonic acid, Phosphatidylcholine (C18:2,C20:4), Phosphatidylcholine (C18:0/C18:2) all of which are decreased.

Alternatively, the said reference may be, preferably, be derived from asubject which has not been brought into contact with Phenobarbitalsodium, Aroclor 1254, Pentachlorobenzene, Beta-ionone, Ethyl-benzene orVinclozoline or a subject known to not suffer from liver enzymeinduction or may be a calculated reference for the analytes in apopulation of subjects. If such a reference is used, identical orsimilar amounts for the metabolites in the test sample and the referenceare indicative for a substance useful for treating liver enzymeinduction.

The term “substance for treating liver enzyme induction” refers tocompounds which may directly interfere with the biological mechanismseliciting liver enzyme induction referred to elsewhere in thisspecification. Accordingly, the activity of the enzymes involved inconjugation and/or oxidation may be modulated. Alternatively, it isenvisaged that substances may affect these activities indirectly by,e.g., modulating the expression of the enzymes or other factors involvedin the said processes. Substances to be identified by the method of thepresent invention may be organic and inorganic chemicals, such as smallmolecules, polynucleotides, oligonucleotides, peptides, polypeptidesincluding antibodies or other artificial or biological polymers.Preferably, the substances are suitable as drugs, pro-drugs or leadsubstances for the development of drugs or pro-drugs. Treating as meantherein includes not only a reversion of the pro-pathologically enzymeinduced liver condition to a physiological condition wherein the enzymesare within their physiological abundance and activities (i.e. equal orless than their upper limits of normal) but also amelioration, i.e. adecrease of the enzymatic induction in the liver. Treating should,preferably, affect at least a significant cohort of individuals of apopulation which is subjected thereto but must not successfully occur inall of the individuals subjected to the said treatment.

It is to be understood that if the methods of the present invention areto be used for identifying drugs for the therapy of liver enzymeinduction or for toxicological assessments of compounds (i.e.determining whether a compound is capable of inducing liver enzymeinduction), test samples of a plurality of subjects may be investigatedfor statistical reasons. Preferably, the metabolome within such a cohortof test subjects shall be as similar as possible in order to avoiddifferences which are caused, e.g., by factors other than the compoundto be investigated. Subjects to be used for the said methods are,preferably, laboratory animals such as rodents and more preferably rats.It is to be understood further that the said laboratory animals shallbe, preferably, sacrificed after completion of the method of the presentinvention. All subjects of a cohort test and reference animals shall bekept under identical conditions to avoid any differential environmentalinfluences. Suitable conditions and methods of providing such animalsare described in detail in WO2007/014825. Said conditions are herebyincorporated by reference.

The present invention also relates to a data collection comprisingcharacteristic values for the following analytes Stearic acid (C18:0),Galactose, Lignoceric acid (C24:0), Behenic acid (C22:0), Nervonic acid(C24:1), 3- and 5-Methoxysphingosine, Cholesterol, Threonic acid,Phosphatidylcholine (C18:0/C18:2),1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine, Eicosapentanoic acid,Phosphatidylcholine (C18:0/C22:6) and/or at least the following analytesGlycerol, Palmitic acid (C16:0), Linoleic acid (C18:cis[9,12]2), Stearicacid (C18:0), Arachidonic acid (C20:cis-[5,8,11,14]4), Docosahexaenoicacid (C22:cis[4,7,10,13,16,19]6), Cholesterol, Glycerolphosphate,Galactose, Lignoceric acid (C24:0), Dodecanol, Heptadecanoic acid(C17:0), Eicosanoic acid (C20:0), myo-Inositol-2-monophosphate, Behenicacid (C22:0), Nervonic acid (C24:1), gamma-Linolenic acid(C18:cis[6,9,12]3), 3- and 5-Methoxysphingosine, Threonic acid,Phosphatidylcholine (C18:2, C20:4), Phosphatidylcholine (C18:0/C18:2).

The term “data collection” refers to a collection of data which may bephysically and/or logically grouped together. Accordingly, the datacollection may be implemented in a single data storage medium or inphysically separated data storage media being operatively linked to eachother. Preferably, the data collection is implemented by means of adatabase. Thus, a database as used herein comprises the data collectionon a suitable storage medium. Moreover, the database, preferably,further comprises a database management system. The database managementsystem is, preferably, a network-based, hierarchical or object-orienteddatabase management system. Furthermore, the database may be a federalor integrated database. More preferably, the database will beimplemented as a distributed (federal) system, e.g. as aClient-Server-System. More preferably, the database is structured as toallow a search algorithm to compare a test data set with the data setscomprised by the data collection. Specifically, by using such analgorithm, the database can be searched for similar or identical datasets being indicative for liver enzyme induction (e.g. a query search).Thus, if an identical or similar data set can be identified in the datacollection, the test data set will be associated with liver enzymeinduction properties. Consequently, the information obtained from thedata collection can be used to diagnose liver enzyme induction based ona test data set obtained from a subject.

Moreover, the present invention pertains to a data storage mediumcomprising the said data collection.

The term “data storage medium” as used herein encompasses data storagemedia which are based on single physical entities such as a CD, aCD-ROM, a hard disk, optical storage media, or a diskette. Moreover, theterm further includes data storage media consisting of physicallyseparated entities which are operatively linked to each other in amanner as to provide the aforementioned data collection, preferably, ina suitable way for a query search.

The present invention also relates to a system comprising

-   -   (a) means for comparing characteristic values of analytes of a        sample operatively linked to    -   (b) the data storage medium of the present invention.

The term “system” as used herein relates to different means which areoperatively linked to each other. Said means may be implemented in asingle device or may be implemented in physically separated deviceswhich are operatively linked to each other. The means for comparingcharacteristic values of analytes operate, preferably, based on analgorithm for comparison as mentioned before. The data storage medium,preferably, comprises the aforementioned data collection or database,wherein each of the stored data sets being indicative for liver enzymeinduction. Thus, the system of the present invention allows identifyingwhether a test data set is comprised by the data collection stored inthe data storage medium. Consequently, the system of the presentinvention may be applied as a diagnostic means in diagnosing liverenzyme induction.

In a preferred embodiment of the system, means for determiningcharacteristic values of metabolites of a sample are comprised.

The term “means for determining characteristic values of analytes”preferably relates to the aforementioned devices for the determinationof analytes such as mass spectrometry devices, NMR devices or devicesfor carrying out chemical or biological assays for the analytes.

The present invention also encompasses a diagnostic compositioncomprising at least one, preferably, at least five of the followinganalytes Stearic acid (C18:0), Galactose, Lignoceric acid (C24:0),Behenic acid (C22:0), Nervonic acid (C24:1), 3- and5-Methoxysphingosine, Cholesterol, Threonic acid, Phosphatidylcholine(C18:0/C18:2), 1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine,Eicosapentanoic acid, Phosphatidylcholine (C18:0/C22:6) and/or at leastone, preferably, at least five of the following analytes Glycerol,Palmitic acid (C16:0), Linoleic acid (C18:cis[9,12]2), Stearic acid(C18:0), Arachidonic acid (C20:cis-[5,8,11,14]4), Docosahexaenoic acid(C22:cis[4,7,10,13,16,19]6), Cholesterol, Glycerolphosphate, Galactose,Lignoceric acid (C24:0), Dodecanol, Heptadecanoic acid (C17:0),Eicosanoic acid (C20:0), myo-Inositol-2-monophosphate, Behenic acid(C22:0), Nervonic acid (C24:1), gamma-Linolenic acid (C18:cis[6,9,12]3),3- and 5-Methoxysphingosine, Threonic acid, Phosphatidylcholine (C18:2,C20:4), Phosphatidylcholine (C18:0/C18:2) or means for the determinationthereof.

Further encompassed by this invention is a diagnostic device comprising

-   -   (a) means for determining characteristic values of at least one,        preferably, at least five of the following analytes Stearic acid        (C18:0), Galactose, Lignoceric acid (C24:0), Behenic acid        (C22:0), Nervonic acid (C24:1), 3- and 5-Methoxysphingosine,        Cholesterol, Threonic acid, Phosphatidylcholine (C18:0/C18:2),        1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine, Eicosapentanoic        acid, Phosphatidylcholine (C18:0/C22:6) and/or at least one,        preferably, at least five of the following analytes Glycerol,        Palmitic acid (C16:0), Linoleic acid (C18:cis[9,12]2), Stearic        acid (C18:0), Arachidonic acid (C20:cis-[5,8,11,14]4),        Docosahexaenoic acid (C22:cis[4,7,10,13,16,19]6), Cholesterol,        Glycerolphosphate, Galactose, Lignoceric acid (C24:0),        Dodecanol, Heptadecanoic acid (C17:0), Eicosanoic acid (C20:0),        myo-Inositol-2-monophosphate, Behenic acid (C22:0), Nervonic        acid (C24:1), gamma-Linolenic acid (C18:cis[6,9,12]3), 3- and        5-Methoxysphingosine, Threonic acid, Phosphatidylcholine (C18:2,        C20:4), Phosphatidylcholine (C18:0/C18:2); and    -   (b) means for diagnosing liver enzyme induction based on the        characteristic values determined by the means of (a).

The term “diagnostic means”, preferably, relates to a diagnostic device,system or biological or chemical assay as specified elsewhere in thedescription in detail.

The expression “means for determining characteristic values of a groupof analytes” refers to devices or agents which are capable ofspecifically recognizing the metabolite(s). Suitable devices may bespectrometric devices such as mass spectrometry, NMR devices or devicesfor carrying out chemical or biological assays for the metabolites.Suitable agents may be compounds which specifically detect themetabolites. Detection as used herein may be a two-step process, i.e.the compound may first bind specifically to the metabolite to bedetected and subsequently generate a detectable signal, e.g.,fluorescent signals, chemiluminescent signals, radioactive signals andthe like. For the generation of the detectable signal, further compoundsmay be required which are all comprised by the term “means fordetermining characteristic values of a group of analytes”. Compoundswhich specifically bind to the metabolite are described elsewhere in thespecification in detail and include, preferably, enzymes, antibodies,ligands, receptors or other biological molecules or chemicals whichspecifically bind to the analytes.

All references referred to above are herewith incorporated by referencewith respect to their entire disclosure content as well as theirspecific disclosure content explicitly referred to in the abovedescription.

The figures show:

FIG. 1: Fragmentation pattern of Phosphatidylcholine (C18:0/C18:2).

FIG. 2: Fragmentation pattern of Phosphatidylcholine (C18:0/C22:6).

The following Examples are merely for the purposes of illustrating thepresent invention. They shall not be construed, whatsoever, to limit thescope of the invention in any respect.

EXAMPLE Biomarkers Associated with Liver Enzyme Induction

A group of each 5 male and female rats was dosed once daily with theindicated compounds (see tables below) at 10 and 100 mg/kg body weightper gavage over 28 days. Additional groups of each 5 male and femaleanimals served as controls. Before starting the treatment period,animals, which were 62-64 days old when supplied, were acclimatized tothe housing and environmental conditions for 7 days. All animals of theanimal population were kept under the same constant temperature(20-24±3° C.) and the same constant humidity (30-70%). Each animal ofthe animal population was kept in a separate cage. The animals of theanimal population are fed ad libitum. The food to be used was beessentially free of chemical or microbial contaminants. Drinking waterwas also offered ad libitum. Accordingly, the water was be free ofchemical and microbial contaminants as laid down in the EuropeanDrinking Water Directive 98/83/EG. The illumination period was 12 hourslight followed by 12 hours darkness (12 hours light, from 6:00 to 18:00,and 12 hours darkness, from 18:00 to 6:00).

In the morning of day 7, 14, and 28, blood was taken from theretroorbital venous plexus from fasted anaesthetized animals. From eachanimal, 1 ml of blood was collected with EDTA as anticoagulant. Thesamples were be centrifuged for generation of plasma. All plasma sampleswere covered with a N₂ atmosphere and then stored at—80° C. untilanalysis.

For mass spectrometry-based metabolite profiling analyses plasma sampleswere extracted and a polar and a non-polar fraction was obtained. ForGC-MS analysis, the non-polar fraction was treated with methanol underacidic conditions to yield the fatty acid methyl esters. Both fractionswere further derivatised with O-methyl-hydroxyamine hydrochloride andpyridine to convert oxo-groups to O-methyloximes and subsequently with asilylating agent before analysis. In LC-MS analysis, both fractions werereconstituted in appropriate solvent mixtures. HPLC was performed bygradient elution on reversed phase separation columns. For massspectrometric detection metanomics proprietary technology was appliedwhich allows target and high sensitivity MRM (Multiple ReactionMonitoring) profiling in parallel to a full screen analysis.

Following comprehensive analytical validation steps, the data for eachanalyte were normalized against data from pool samples. These sampleswere run in parallel through the whole process to account for processvariability. The significance of treatment group values specific forsex, dose group and metabolite was determined by comparing means of thetreated groups to the means of the respective untreated control groupsusing Student's t-test. Normalized treatment group values and theirsignificance were fed into a database for further statistics and datamining processes.

The changes of the group of plasma metabolites being indicative forliver enzyme induction after treatment of the rats are shown in thefollowing tables:

TABLE 1 Markers for liver enzyme induction in male ratsPentachlorobenzene Aroclor 1254 (MOA12) (MOA61) Metabolite Direction mh7mh14 mh28 mh7 mh14 mh28 Stearic acid (C18:0) up 1.34 1.25 1.05 1.50 1.651.69 Galactose, lipid fraction up 1.37 1.37 1.51 1.44 1.41 1.61Lignoceric acid (C24:0) up 1.32 1.16 1.21 1.40 1.29 1.28 Behenic acid(C22:0) up 1.43 1.26 1.34 1.27 1.56 1.40 Nervonic acid (C24:1) up 1.301.78 1.17 1.23 1.56 1.64 Sphingolipids (3- and 5-Methoxysphingosine) up1.42 1.79 1.46 1.52 1.99 2.07 Cholesterol up 1.31 1.46 1.04 1.14 1.721.43 Threonic acid up 3.06 2.98 3.48 1.68 1.82 2.90 Phosphatidylcholine(C18:0/C18:2) up 1.01 1.11 1.10 1.10 1.15 1.141,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine up 1.27 1.35 1.27 1.271.40 1.35 Eicosapentanoic acid up 1.4 1.4 1.4 1.4 1.4 1.4Phosphatidylcholine (C18:0/C22:6) up 1.12 1.15 1.06 1.20 1.23 1.12

TABLE 2 Markers for liver enzyme induction in female ratsPentachlorobenzene Aroclor 1254 (MOA12) (MOA61) Metabolite Direction fh7fh14 fh28 fh7 fh14 fh28 Glycerol, lipid fraction up 2.38 7.37 2.68 1.011.24 1.16 Palmitic acid (C16:0) up 1.59 3.46 1.86 1.16 1.27 1.19Linoleic acid (C18:cis[9,12]2) up 2.11 5.23 2.69 1.34 1.34 1.54 Stearicacid (C18:0) up 1.30 1.87 1.65 1.34 1.54 1.91 Arachidonic acid(C20:cis-[5,8,11,14]4) up 1.27 1.89 1.50 1.25 1.48 1.53 Docosahexaenoicacid (C22:cis[4,7,10,13,16,19]6) up 1.79 2.49 1.57 1.21 1.66 1.45Cholesterol up 1.23 1.64 1.62 1.43 1.45 1.53 Glycerolphosphate, lipidfraction up 1.27 1.52 1.43 1.31 1.68 1.89 Galactose, lipid fraction up1.10 1.28 1.40 1.10 1.60 1.38 Lignoceric acid (C24:0) up 1.39 1.60 1.751.07 1.40 1.18 Dodecanol up 1.17 1.79 1.40 1.31 1.27 1.26 Heptadecanoicacid (C17:0) up 1.32 1.89 1.54 1.39 1.10 1.40 Eicosanoic acid (C20:0) up1.13 2.76 1.98 1.87 2.25 1.40 Tricosanoic acid (C23:0) up 1.18 1.46 1.451.01 1.22 1.13 myo-Inositol-2-monophosphate, lipid fraction (m... up1.81 3.61 1.93 2.01 1.91 1.23 Behenic acid (C22:0) up 1.30 1.60 1.411.57 1.27 1.24 Nervonic acid (C24:1) up 0.97 1.55 1.46 1.20 1.13 1.31gamma-Linolenic acid (C18:cis[6,9,12]3) up 1.71 4.53 2.98 1.61 1.26 1.33Sphingolipids (3- and 5-Methoxysphingosine) up 1.43 1.77 1.73 1.28 2.011.67 Threonic acid up 3.67 3.63 4.57 2.11 1.87 2.00 Phosphatidylcholine(C18:0/C18:2) up 1.14 1.20 1.23 1.16 1.13 1.151,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine up 1.28 1.72 1.51 1.241.30 1.16

1-22. (canceled)
 23. A method for diagnosing liver enzyme induction as apredisposition for liver toxicity and diseases or disorders accompaniedtherewith comprising: (a) determining the amount of at least five of thefollowing analytes Stearic acid (C18:0), Galactose, Lignoceric acid(C24:0), Behenic acid (C22:0), Nervonic acid (C24:1), 3- and5-Methoxysphingosine, Cholesterol, Threonic acid, Phosphatidylcholine(C18:0/C18:2), 1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine,Eicosapentanoic acid, Phosphatidylcholine (C18:0/C22:6) in a testbodyfluid sample of a male subject suspected to suffer from liver enzymeinduction or at least five of the following analytes Glycerol, Palmiticacid (C16:0), Linoleic acid (C18:cis[9,12]2), Stearic acid (C18:0),Arachidonic acid (C20:cis-[5,8,11,14]4), Docosahexaenoic acid(C22:cis[4,7,10,13,16,19]6), Cholesterol, Glycerolphosphate, Galactose,Lignoceric acid (C24:0), Dodecanol, Heptadecanoic acid (C17:0),Eicosanoic acid (C20:0), myo-Inositol-2-monophosphate, Behenic acid(C22:0), Nervonic acid (C24:1), gamma-Linolenic acid (C18:cis[6,9,12]3),3- and 5-Methoxysphingosine, Threonic acid, Phosphatidylcholine (C18:2,C20:4), Phosphatidylcholine (C18:0/C18:2) in a test bodyfluid sample ofa female subject suspected to suffer from liver enzyme induction as apredisposition for liver toxicity and diseases or disorders accompaniedtherewith, and (b) comparing the amounts determined in step (a) to areference, whereby liver enzyme induction as a predisposition for livertoxicity and diseases or disorders accompanied therewith is to bediagnosed.
 24. The method of claim 23, wherein said subject has beenbrought into contact with a compound suspected to be capable of inducingliver enzyme induction as a predisposition for liver toxicity anddiseases or disorders accompanied therewith.
 25. A method of determiningwhether a compound is capable of inducing liver enzyme induction as apredisposition for liver toxicity and diseases or disorders accompaniedtherewith in a subject comprising: (a) determining in a test bodyfluidsample of a male subject which has been brought into contact with acompound suspected to be capable of inducing liver enzyme induction theamount of at least five of the following analytes Stearic acid (C18:0),Galactose, Lignoceric acid (C24:0), Behenic acid (C22:0), Nervonic acid(C24:1), 3- and 5-Methoxysphingosine, Cholesterol, Threonic acid,Phosphatidylcholine (C18:0/C18:2),1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine, Eicosapentanoic acid,Phosphatidylcholine (C18:0/C22:6) or in a test bodyfluid sample of afemale which has been brought into contact with a compound suspected tobe capable of inducing liver enzyme induction at least five of thefollowing analytes Glycerol, Palmitic acid (C16:0), Linoleic acid(C18:cis[9,12]2), Stearic acid (C18:0), Arachidonic acid(C20:cis-[5,8,11,14]4), Docosahexaenoic acid(C22:cis[4,7,10,13,16,19]6), Cholesterol, Glycerolphosphate, Galactose,Lignoceric acid (C24:0), Dodecanol, Heptadecanoic acid (C17:0),Eicosanoic acid (C20:0), myo-Inositol-2-monophosphate, Behenic acid(C22:0), Nervonic acid (C24:1), gamma-Linolenic acid (C18:cis[6,9,12]3),3- and 5-Methoxysphingosine, Threonic acid, Phosphatidylcholine (C18:2,C20:4), Phosphatidylcholine (C18:0/C18:2); and (b) comparing the amountsdetermined in step (a) to a reference, whereby the capability of thecompound to induce liver enzyme induction as a predisposition for livertoxicity and diseases or disorders accompanied therewith is determined.26. The method of claim 24, wherein said compound is at least onecompound selected from the group consisting of: Phenobarbital sodium,Aroclor 1254, Pentachlorobenzene, Beta-ionone, Ethyl-benzene orVinclozoline.
 27. The method of claim 23, wherein said reference isderived from (i) a subject which suffers from liver enzyme induction asa predisposition for liver toxicity and diseases or disordersaccompanied therewith or (ii) a subject which has been brought intocontact with at least one compound selected from the group consistingof: Phenobarbital sodium, Aroclor 1254, Pentachlorobenzene, Beta-ionone,Ethyl-benzene or Vinclozoline.
 28. The method of claim 27, whereinidentical amounts for the analytes in the test bodyfluid sample and thereference are indicative for liver enzyme induction as a predispositionfor liver toxicity and diseases or disorders accompanied therewith. 29.The method of claim 23, wherein said reference is derived from (i) asubject known to not suffer from liver enzyme induction as apredisposition for liver toxicity and diseases or disorders accompaniedtherewith or (ii) a subject which has not been brought into contact withat least one compound selected from the group consisting of:Phenobarbital sodium, Aroclor 1254, Pentachlorobenzene, Beta-ionone,Ethyl-benzene or Vinclozoline.
 30. The method of claim 23, wherein saidreference is a calculated reference for the analytes for a population ofsubjects.
 31. The method of claim 29, wherein amounts for the analyteswhich differ in the test sample in comparison to the reference areindicative for liver enzyme induction as a predisposition for livertoxicity and diseases or disorders accompanied therewith.
 32. The methodof claim 29, wherein indicative for liver enzyme induction as apredisposition for liver toxicity and diseases or disorders accompaniedtherewith are amounts of the analytes in comparison to the referencewhich differ as follows: (i) in a test bodyfluid sample of a male:Stearic acid (C18:0), Galactose, Lignoceric acid (C24:0), Behenic acid(C22:0), Nervonic acid (C24:1), 3- and 5-Methoxysphingosine,Cholesterol, Threonic acid, Phosphatidylcholine (C18:0/C18:2),1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine, Eicosapentanoic acid,Phosphatidylcholine (C18:0/C22:6) all of which are increased and (ii) ina test bodyfluid sample of a female subject: Glycerol, Palmitic acid(C16:0), Linoleic acid (C18:cis[9,12]2), Stearic acid (C18:0),Arachidonic acid (C20:cis-[5,8,11,14]4), Docosahexaenoic acid(C22:cis[4,7,10,13,16,19]6), Cholesterol, Glycerolphosphate, Galactose,Lignoceric acid (C24:0), Dodecanol, Heptadecanoic acid (C17:0),Eicosanoic acid (C20:0), myo-Inositol-2-monophosphate, Behenic acid(C22:0), Nervonic acid (C24:1), gamma-Linolenic acid (C18:cis[6,9,12]3),3- and 5-Methoxysphingosine, Threonic acid, Phosphatidylcholine (C18:2,C20:4), Phosphatidylcholine (C18:0/C18:2) all of which are increased.33. A method of identifying a substance for treating liver enzymeinduction as a predisposition for liver toxicity and diseases ordisorders accompanied therewith comprising the steps of: (a) determiningin a test bodyfluid sample of a male subject suffering from liver enzymeinduction as a predisposition for liver toxicity and diseases ordisorders accompanied therewith which has been brought into contact witha candidate substance suspected to be capable of treating liver enzymeinduction the amount of at least five of the following analytes Stearicacid (C18:0), Galactose, Lignoceric acid (C24:0), Behenic acid (C22:0),Nervonic acid (C24:1), 3- and 5-Methoxysphingosine, Cholesterol,Threonic acid, Phosphatidylcholine (C18:0/C18:2),1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine, Eicosapentanoic acid,Phosphatidylcholine (C18:0/C22:6) or in a test bodyfluid sample of afemale subject suffering from liver enzyme induction as a predispositionfor liver toxicity and diseases or disorders accompanied therewith whichhas been brought into contact with a candidate substance suspected to becapable of treating liver enzyme induction the amount of at least fiveof the following analytes Glycerol, Palmitic acid (C16:0), Linoleic acid(C18:cis[9,12]2), Stearic acid (C18:0), Arachidonic acid(C20:cis-[5,8,11,14]4), Docosahexaenoic acid(C22:cis[4,7,10,13,16,19]6), Cholesterol, Glycerolphosphate, Galactose,Lignoceric acid (C24:0), Dodecanol, Heptadecanoic acid (C17:0),Eicosanoic acid (C20:0), myo-Inositol-2-monophosphate, Behenic acid(C22:0), Nervonic acid (C24:1), gamma-Linolenic acid (C18:cis[6,9,12]3),3- and 5-Methoxysphingosine, Threonic acid, Phosphatidylcholine (C18:2,C20:4), Phosphatidylcholine (C18:0/C18:2); and (b) comparing the amountsdetermined in step (a) to a reference, whereby a substance capable oftreating liver enzyme induction as a predisposition for liver toxicityand diseases or disorders accompanied therewith is to be identified. 34.The method of claim 33, wherein said reference is derived from (i) asubject which suffers from liver enzyme induction as a predispositionfor liver toxicity and diseases or disorders accompanied therewith or(ii) a subject which has been brought into contact with at least onecompound selected from the group consisting of: Phenobarbital sodium,Aroclor 1254, Pentachlorobenzene, Beta-ionone, Ethyl-benzene orVinclozoline.
 35. The method of claim 34, wherein amounts for theanalytes which differ in the test bodyfluid sample and the reference areindicative for a substance capable of treating liver enzyme induction asa predisposition for liver toxicity and diseases or disordersaccompanied therewith.
 36. The method of claim 33, wherein indicativefor a substance capable of treating liver enzyme induction as apredisposition for liver toxicity and diseases or disorders accompaniedtherewith are amounts of the analytes in comparison to the referencewhich differ as follows: (i) in a test bodyfluid sample of a male:Stearic acid (C18:0), Galactose, Lignoceric acid (C24:0), Behenic acid(C22:0), Nervonic acid (C24:1), 3- and 5-Methoxysphingosine,Cholesterol, Threonic acid, Phosphatidylcholine (C18:0/C18:2),1,2-Dioleoyl-sn-glycero-3-phosphatidyl-L-serine, Eicosapentanoic acid,Phosphatidylcholine (C18:0/C22:6) all of which are decreased and (ii) ina test bodyfluid sample of a female subject: Glycerol, Palmitic acid(C16:0), Linoleic acid (C18:cis[9,12]2), Stearic acid (C18:0),Arachidonic acid (C20:cis-[5,8,11,14]4), Docosahexaenoic acid(C22:cis[4,7,10,13,16,19]6), Cholesterol, Glycerolphosphate, Galactose,Lignoceric acid (C24:0), Dodecanol, Heptadecanoic acid (C17:0),Eicosanoic acid (C20:0), myo-Inositol-2-monophosphate, Behenic acid(C22:0), Nervonic acid (C24:1), gamma-Linolenic acid (C18:cis[6,9,12]3),3- and 5-Methoxysphingosine, Threonic acid, Phosphatidylcholine (C18:2,C20:4), Phosphatidylcholine (C18:0/C18:2) all of which are decreased.37. The method of claim 33, wherein said reference is derived from (i) asubject known to not suffer from liver enzyme induction as apredisposition for liver toxicity and diseases or disorders accompaniedtherewith or (ii) a subject which has not been brought into contact withat least one compound selected from the group consisting of:Phenobarbital sodium, Aroclor 1254, Pentachlorobenzene, Beta-ionone. 38.The method of claim 33, wherein said reference is a calculated referencefor the analytes in a population of subjects.
 39. The method of claim37, wherein identical amounts for the analytes in the test bodyfluidsample and the reference are indicative for a substance capable oftreating liver enzyme induction as a predisposition for liver toxicityand diseases or disorders accompanied therewith.
 40. The method of claim23, wherein said liver enzyme induction is indicative for apredisposition for at least one disorder or disease selected from thegroup consisting of: liver cell necrosis, hepatitis, steatosis,cirrhosis, phospholipoidosis, cholestasis, cholangitis, thrombosis ofliver veins, liver tumors.
 41. The method of claim 23, wherein saiddetermining the group of analytes comprises mass spectrometry (MS). 42.The method of claim 41, wherein said mass spectrometry is liquidchromatography (LC)-MS or gas chromatography (GC)-MS.
 43. The method ofclaim 23, wherein said body fluid is blood.
 44. The method of claim 23,wherein said subject is a mammal.